A novel Phosphoinositide-specific phospholipase C (PI-PLC) has been described in T. cruzi, the etiologic agent of Chagas' disease. This enzyme possesses an N-myristoylation and palmitoylation consensus sequence that had not been described previously in any other PI-PLC from eukaryotic cells. It has been confirmed that the enzyme is myristoylated and palmitoylated. Recently, we demonstrated that there is a correlation between the expression levels of the TcPI-PLC and the differentiation of trypomastigotes into amastigotes. The overexpression of TcPI-PLC in the plasma membrane stimulated differentiation and reduction in the TcPI-PLC expression inhibited the process. In addition, preliminary evidence showed that the enzyme could be involved in shedding of Ssp-4, a GPI-anchored protein containing inositolphosphoceramide in its lipid anchor. This was suggested by the simultaneous localization of TcPI- PLC and Ssp-4 in the external surface of the cells, the ability of the TcPI-PLC to hydrolyze inositolphosphoceramide in vitro, the shedding of Ssp-4 without its lipid anchor (as demonstrated by its cross-reactive determinant (CRD) reactivity), and the increase in cellular ceramide when maximal surface expression of TcPI-PLC takes place. Ceramide is also an important second messenger involved in cellular differentiation. Based on all these findings our hypothesis is that TcPI-PLCs could be responsible for multiple functions as it travels to the outer surface of the cells: (1) hydrolysis of PIP2 and generation of IPS in the parasites, this effect being important for their differentiation; (2) hydrolysis of the glycoinositolphospholipids of GPI-anchors of parasite glycoproteins, which results in shedding of proteins to the medium; and (3) hydrolysis of PIP2 from the host cells leading to changes in its cytoskeleton and generation of IPSthat could be involved in cell signaling in the host. According to these findings, the specific aims of the proposal are: (1) To investigate the role of fatty acid modifications in TcPI-PLC localization and regulation of membrane binding; (2) To investigate whether TcPI-PLC is involved in the stress response of the parasite, in the hydrolysis of parasite and mammalian phospholipids, and its importance for cell differentiation and host- parasite interactions; (3) To investigate the transport mechanism of TcPI-PLC to the outer surface of the cells.